Engineered in vivo nuclear fusion: application to regenerative medicine. This application addresses broad Challenge Area (11) Regenerative Medicine and specific Challenge Topic, 11-DK-104: Use of Hematopoietic Stem Cells (HSC) to regenerate or repair mesenchymal tissues. This application focuses on the development of a new cell-based strategy for regenerative medicine and gene therapy which depends upon the ability to genetically engineer hematopoietic stem cells or other cell populations to be capable of efficient fusion to cells within organs and tissues after their transplantation in vivo, using murine retroviral envelope or human viral-like fusogenic gene products. The approach seeks to build upon provocative recent studies of the ability of hematopoietic or other stem cells to transdifferentiate into unexpected differentiated cells which have suggested that spontaneous cell fusion involving circulating hematopoietic cells and cells within organs and tissues can occur, particularly in response to tissue injury, and may represent an important mechanism for the repair of organs and tissues. The hypothesis underlying this grant application is that genetic technologies can provide a means of dramatically increasing the efficiency of cell fusion of transplanted cells, and that such engineered cell-cell fusion in vivo can provide a broad and powerful platform for repairing tissues and for the delivery of useful therapeutics. A major goal of the two-year program is to rapidly assess the potential breadth of the new technology so that subsequent studies can immediately focus on the most promising therapeutic applications. In a series of in vitro experiments, effort will be made to further understand the experimental parameters governing engineered cell-cell fusion via fusogenic viral envelope gene products, with an emphasis on characterizing the viability and fate of fused cells, and determining how expression levels of either the fusogenic envelopes or their corresponding receptors affect the absolute efficiency of fusion and the average number of nuclei that are fused. In vivo studies will focus on a determination of the range of tissues and organs that are amenable to cell fusion after local or systemic delivery of different types of donor cells. In addition to determining the efficiency of nuclear transfer to muscle and other tissues and organs, the capacity of donor nuclei derived from different cell types to provide for the expression of therapeutic gene products, and the immunological consequences of the transplantation of allogeneic and xenogeneic cells, will be determined. A particularly important goal of the in vivo studies will be to understand how interactions between donor and recipient nuclei affect the extent of reprogramming and/or maintenance of the differentiated phenotype of the donor and recipient nuclei in different tissues. Of specific interest is whether in vivo nuclear fusion can be employed to reprogram cells in the pancreas or liver to the islet phenotype, and/or to enable the preservation of the specialized differentiated functions of specific cells, such as insulin or antibody secreting cells via introduction of nuclei from those specialized cells into muscle or other tissues. In efforts to systemically deliver nuclei to tissues via the transplantation of hematopoietic stem cells, an important objective is to directly determine whether the expression of fusogenic gene products can provide for increased levels of engraftment of donor derived nuclei and regenerative repair relative to untransduced cells. PUBLIC HEALTH RELEVANCE: The proposed research is highly relevant to human health, as the goal of the studies is to develop a novel cell transplantation approach to regenerative medicine. Such a therapy could have a major impact upon the treatment of many inherited and acquired diseases for which there are currently no effective treatments